Wojcik Radoslaw, Baranowski Marek R, Markiewicz Lukasz, Kubacka Dorota, Bednarczyk Marcelina, Baran Natalia, Wojtczak Anna, Sikorski Pawel J, Zuberek Joanna, Kowalska Joanna, Jemielity Jacek
Centre of New Technologies, University of Warsaw, 02097 Warsaw, Poland.
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02093 Warsaw, Poland.
Pharmaceutics. 2021 Nov 16;13(11):1941. doi: 10.3390/pharmaceutics13111941.
Dinucleotide analogs of the messenger RNA cap (mGpppN) are useful research tools and have potential applications as translational inhibitors or reagents for modification of in vitro transcribed mRNAs. It has been previously reported that replacing the methyl group at the N7-position with benzyl (Bn) produces a dinucleotide cap with superior properties. Here, we followed up on this finding by synthesizing 17 novel BnGpppG analogs and determining their structure-activity relationship regarding translation and translational inhibition. The compounds were prepared in two steps, including selective N7-alkylation of guanosine 5'-monophosphate by arylmethyl bromide followed by coupling with imidazole-activated GDP, with total yields varying from 22% to 62%. The compounds were then evaluated by determining their affinity for eukaryotic translation initiation factor 4E (eIF4E), testing their susceptibility to decapping pyrophosphatase, DcpS-which is most likely the major cellular enzyme targeting this type of compound-and determining their translation inhibitory properties in vitro. We also synthesized mRNAs capped with the evaluated compounds and tested their translational properties in A549 cells. Our studies identified N7-(4-halogenbenzyl) substituents as promising modifications in the contexts of either mRNA translation or translational inhibition. Finally, to gain more insight into the consequences at the molecular level of N7-benzylation of the mRNA cap, we determined the crystal structures of three compounds with eIF4E.
信使核糖核酸帽（mGpppN）的二核苷酸类似物是有用的研究工具，并且作为翻译抑制剂或用于修饰体外转录mRNA的试剂具有潜在应用。先前已有报道称，用苄基（Bn）取代N7位的甲基可产生具有优异性能的二核苷酸帽。在此，我们通过合成17种新型BnGpppG类似物并确定它们在翻译和翻译抑制方面的构效关系，对这一发现进行了跟进研究。这些化合物通过两步制备，包括用芳基甲基溴对5'-单磷酸鸟苷进行选择性N7-烷基化，然后与咪唑活化的GDP偶联，总产率在22%至62%之间。然后通过测定它们对真核翻译起始因子4E（eIF4E）的亲和力、测试它们对去帽焦磷酸酶DcpS的敏感性（DcpS很可能是靶向这类化合物的主要细胞酶）以及在体外确定它们的翻译抑制特性来评估这些化合物。我们还合成了用评估的化合物加帽的mRNA，并在A549细胞中测试了它们的翻译特性。我们的研究确定N7-（4-卤代苄基）取代基在mRNA翻译或翻译抑制方面是有前景的修饰。最后，为了更深入了解mRNA帽的N7-苄基化在分子水平上的后果，我们测定了三种化合物与eIF4E的晶体结构。
